Detection of Ralstonia solanacearum by loop-mediated isothermal amplification.
Ralstonia solanacearum is a pathogenic bacterium that causes wilt in over 200 plant species. Here we report a rapid and sensitive detection of R. solanacearum using an isothermal method for copying DNA known as loop-mediated amplification (LAMP). A set of four primers was designed to replicate the gene coding for the flagellar subunit, fliC, and conditions for detection were optimized to complete in 60 min at 65 degrees C. Magnesium pyrophosphate resulting from the amplification reaction could be detected optically as an increase in the solution turbidity, and the DNA products spread in a reproducible ladder-like banding pattern after electrophoresis in an agarose gel. Replication of the fliC gene was detected only from R. solanacearum. The detection limit of this LAMP assay was between 10(4) to 10(6) colony forming units/ml, and the technique may be useful for developing rapid and sensitive detection methods for the R. solanacearum pathogen in soil and water.
Kubota
R
R
Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu 96822, USA.
Vine
B G
BG
Alvarez
A M
AM
Jenkins
D M
DM
D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D043368:Ralstonia solanacearum / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial